Conditional-lethal temperature-sensitive (ts) mutants of human adenovirus 2 will be constructed in vitro by mutagenizing specific restriction fragments of the viral DNA and joining with the rest of the genome by (1) in vitro recombination (i.e. ligation) of restriction endonuclease-generated fragments (2) construction of a heteroduplex with immutagenized DNA and (3) co-transfection with immutagenized wild type or host range mutant DNAs. The DNA will be transfected into a human embryonic kidney cell line transformed by sheared Ad 5 DNA (293 cells). The progeny virus will be screened for ts mutants in KB cells. Ts mutants defective in early viral genes will be generated by specific mutagenization of the left and right ends of the genome. These mutants will be characterized with regard to transformation of nonpermissive rat cells, viral DNA synthesis, regulation of expression of viral genes, synthesis of viral polypeptides and assembly of viral particles.